Luteinizing hormone-releasing hormone (LHRH) is a major regulator of reproduction in mammals. This decapeptide is processed from prohormone, pro-LHRH. At least four different enzymatic steps are involved in the conversion of pro-LHRH to LHRH. One of these processing enzymes may be carboxypeptidase E (CPE). Recently, a mouse line was identified with a point mutation in CPE. These CPEfat mice are obese, diabetic, and infertile. The overall objective of the proposal is to determine whether CPE is involved in processing the pro-LHRH in vivo and whether a defect in this enzyme is responsible for the infertility in the CPEfat mouse. Aim I: There are four aspects to this first specific aim. First, processing of pro-LHRH will be examined in hypothalami from male and female wild type, heterozygous, and homozygous CPEfat mice. These data should identify which steps in pro-LHRH processing are deficient in the homozygotes. Second, LHRH gene expression and pro-LHRH biosynthesis will be compared among the groups of mice. Third, since pro-LHRH processing may involve three different enzymes besides CPE, expression of these enzymes will also be studied. Fourth, biological activity of the various pro-LHRH intermediates will be assessed in dispersed anterior pituitary cultures from these groups of mice. Finally, the hypothalamus, pituitary and gonads will be evaluated to determine whether the mutation in CPE affects additional responses from these organs. Aim II: A transgenic approach will be used to rescue the CPEfat phenotype and restore processing of the pro-LHRH to LHRH. In this case, mice will be genetically targeted for CPE expression in hypothalamic LHRH neurons. These transgenic mice will be bred with heterozygous CPEfat mice to produce a mouse that is homozygous for the CPEfat mutation, but has CPE selectively expressed in LHRH neurons. The abilities of these mice to process pro-LHRH to LHRH and responses from the gonads, pituitary and hypothalamus will be evaluated as in Aim I. Aim III: Mice homozygous for the CPEfat mutation that have selective expression of CPE in LHRH neurons will be evaluated for their abilities to reproduce. This series of experiments in the CPEfat mouse presents us with the unique opportunity to clearly link a deficiency in pro-LHRH processing with a defect in reproduction. The biochemical, physiological, and gene-targeting principles derived from this work should broaden our understanding of basic mechanisms that regulate reproduction in mammals and it may serve as a useful example for repairing genetically-based diseases or other defects in the future.